A major hurdle to developing disease-altering therapies for Parkinson?s disease (PD) has been a gap in knowledge of the molecular mechanisms underlying the loss of dopaminergic neurons in the midbrains of PD patients. Multiple lines of evidence suggest that aggregation of the presynaptic protein alpha-synuclein (aSyn) at membrane surfaces plays a central role in neuronal cell death in PD. Recent published findings indicate that the protein endosulfine-alpha (ENSA), previously shown to interact selectively with lipid-bound aSyn, interferes with membrane-induced aSyn aggregation and aSyn neurotoxicity, whereas a mimic of ENSA phosphorylated at residue S109 by protein kinase A has no inhibitory activity. Moreover, ENSA was found to be down- regulated in the brains of synucleinopathy patients. However, it is unknown whether ENSA down-regulation promotes nigral dopaminergic cell death, or whether ENSA undergoes changes in S109 phosphorylation in PD brain. Resolving these issues is essential to establish a strong scientific premise for developing therapies aimed at enhancing ENSA neuroprotective activity by modulating the protein?s expression and/or phosphorylation. The long-term goal of this research is to define the underlying molecular mechanisms that contribute to neurodegeneration in PD patients. The overall objective in this application is to determine the effects of ENSA down-regulation and phosphorylation on aSyn neurotoxicity. The rationale for this research is that its successful completion would provide a strong evidence-based foundation to justify screening for agents that up-regulate ENSA expression or inhibit ENSA phosphorylation as new therapeutic candidates for PD. The central hypothesis, formulated on the basis of extensive preliminary data, is that ENSA in its unphosphorylated form interferes with membrane-induced aSyn aggregation and aSyn neurotoxicity in the brains of PD patients. This hypothesis will be addressed with the following specific aims: (1) Determine the effect of ENSA knockdown on aSyn neurotoxicity; and (2) Determine the relative abundance of pS109-ENSA in PD midbrain. Aim 1 studies will involve characterizing rat primary midbrain cultures co-transduced with adenoviruses encoding aSyn and an shRNA specific for rat ENSA in terms of dopaminergic cell viability and neurite lengths. Additional experiments will involve examining rats injected unilaterally in the substantia nigra with rAAV virus encoding an ENSA-targeting shRNA for evidence of motor dysfunction and PD-related neuropathology. Aim 2 studies will involve characterizing homogenates prepared from patient and control midbrain samples in terms of relative levels of ENSA S109 phosphorylation via quantitative LC/MS/MS, and validating the MS data via immunoblotting using a new anti-pS109-ENSA antibody. This approach is innovative because it is focused on new directions related to the role of nigral ENSA in neurodegenerative disease. The research is significant because the new knowledge from this study would set the stage for developing therapeutic strategies to stimulate ENSA-mediated neuroprotective activity in the brains of PD patients.